Autophagy research in the heart has focused almost exclusively on macroautophagy, in which double membrane vesicles transport molecules and organelles to lysosomes for degradation. In contrast, the subject of the parent R01 is a distinct process termed chaperone mediated autophagy (CMA). In CMA, cytoplasmic proteins are selectively targeted for degradation through a mechanism in which Hsc70 and co-chaperones bind a recognition motif on the target protein. This complex then translocates to the lysosome where it is imported into the lumen by LAMP2A, a lysosomal transmembrane protein that is necessary, specific, and rate-limiting for CMA. Our informatics analyses suggest that there are ~7000 potential CMA substrates in the heart. However, there was no means to assess the functional significance of CMA in healthy or diseased hearts until we inactivated CMA in cardiomyocytes by creating inducible, cardiomyocyte-specific LAMP2A knockout mice. While these mice are normal at baseline, an unanticipated phenotype emerges when they are stressed with pressure overload or post-myocardial infarction heart failure: Systolic dysfunction in each of these models is markedly attenuated by inhibition of CMA ? not worsened as one might expect from the traditional role of autophagy in ameliorating cellular stresses. Mechanistic investigations revealed another unexpected relationship: Inhibition of CMA induces mitophagy, a process that maintains the overall health of the mitochondrial pool by eliminating defective organelles. Based on these observations, we proposed a novel paradigm in which CMA, activated in response to cardiac stress, mediates cardiac dysfunction by depleting cardiomyocytes of proteins that would normally promote mitochondrial quality control through mitophagy. In the parent grant, we proposed to test this model and further delineate molecular mechanism. Aim 1 addresses the functional role of CMA in heart failure using both pressure overload and MI models; while Aim 2 seeks to identify CMA substrates that induce mitophagy. One promising candidate is the mitophagy inducer, Parkin, which we believe is a CMA substrate. However, Aim 2 also sought to identify additional candidates using an unbiased approach. This involved first performing lysosomal proteomics to define CMA substrates in cardiomyocytes, following which these proteins would be test for the abilities to induce mitophagy. The research proposed in this diversity supplement addresses the same question, but using a novel approach that was not available at the time the parent R01 was written. This new approach makes use the results of a genome-wide CRISPR/Cas9 screen for novel mitophagy inducers that was conducted by our collaborator Dr. Zoltan Arany. We propose to use informatics (Diversity Supplement Aim 1) and biochemical approaches (Diversity Supplement Aim 2) to identify CMA substrates among the mitophagy inducers revealed by the CRISPR screen. We will then test whether these candidates are responsible for the induction of mitophagy when CMA is inhibited using isolated adult cardiomyocytes (Diversity Supplement Aim 3) and intact mice (Diversity Supplement Aim 4). Thus, the experiments proposed in this diversity supplement complement and accelerate the approach proposed in the parent grant. Taken together, the combination will advance our understanding of this novel pathway in which stress-activated CMA impairs mitophagy and mitochondrial quality control resulting in heart failure.